Hasmik Mkrtchyan

Early Embryonic Chromosome Instability Results in Stable Mosaic Pattern in Human Tissues

The discovery of copy number variations (CNV) in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.

The Human Genome Puzzle - the Role of Copy Number Variation in Somatic Mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.

Complex rearranged small supernumerary marker chromosomes (sSMC), three new cases; evidence for an underestimated entity?

BackgroundSmall supernumerary marker chromosomes (sSMC) are present ~2.6 × 106 human worldwide. sSMC are a heterogeneous group of derivative chromosomes concerning their clinical consequences as well as their chromosomal origin and shape. Besides the sSMC present in Emanuel syndrome, i.e. der(22)t(11;22)(q23;q11), only few so-called complex sSMC are reported.ResultsHere we report three new cases of unique complex sSMC. One was a de novo case with a dic(13 or 21;22) and two were maternally derived: a der(18)t(8;18) and a der(13 or 21)t(13 or 21;18). Thus, in summary, now 22 cases of unique complex sSMC are available in the literature. However, this special kind of sSMC might be under-diagnosed among sSMC-carriers.ConclusionMore comprehensive characterization of sSMC and approaches like reverse fluorescence in situ hybridization (FISH) or array based comparative genomic hybridization (array-CGH) might identify them to be more frequent than only ~0.9% among all sSMC.

First Molecular Cytogenetic High Resolution Characterization of the NIH 3T3 Cell Line by Murine Multicolor Banding

Since being established in 1963, the murine fibroblast cell line NIH 3T3 has been used in thousands of studies. NIH 3T3 immortalized spontaneously and became tetraploid shortly after its establishment. Here we report the first molecular cytogenetic characterization of NIH 3T3 using fluorescence in situ hybridization based multicolor banding (mcb). Overall, a complex rearranged karyotype presenting 16 breakpoints was characterized. Also it was possible to deduce the resulting gains and losses of copy numbers in NIH 3T3. Overall, only 1.8% of the NIH 3T3 genome is disome, 26.2% tri-, 60% tetra-, 10.8% quinta-, and 1.2% hexasome. Strikingly, the cell line gained only 4 derivative chromosomes since its first cytogenetic description in 1989. An attempt to align the observed imbalances of the studied cell line with their homologous regions in humans gave the following surprising result: NIH 3T3 shows imbalances as typically seen in human solid cancers of ectodermal origin.

Heteromorphic variants of chromosome 9

BackgroundHeterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers.ResultsIn this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes.ConclusionsBased on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants.

Molecular Cytogenetic Characterization of the Human Cerebral Microvessel Endothelial Cell Line hCMEC/D3

The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45∼48,XX, –X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13∼14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],–22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.

Banding and molecular cytogenetic studies detected a CBFB-MYH11 fusion gene that appeared as abnormal chromosomes 1 and 16 in a baby with acute myeloid leukemia FAB M4-Eo.

The acute myeloid leukemia (AML) subtype M4Eo occurs in 5% of all AML cases and is usually associated with either an inv(16)(p13.1q22) or a t(16;16)(p13.1;q22) chromosomal abnormality. At the molecular level, these abnormalities generate a CBFB-MYH11 fusion gene. Patients with this genetic alteration are usually assigned to a low-risk group and thus receive standard chemotherapy. AML-M4Eo is rarely found in infants. We describe clinical, conventional banding, and molecular cytogenetic data for a 12-month-old baby with AML-M4Eo and a chimeric CBFB-MYH11 fusion gene masked by a novel rearrangement between chromosomes 1 and 16. This rearrangement characterizes a new type of inv(16)(p13.1q22) masked by a chromosome translocation.

New Immortalized Cell Lines of Patients With Small Supernumerary Marker Chromosome Towards the Establishment of a Cell Bank

Sixteen newly established cell lines with small supernumerary marker chromosomes (sSMC) derived from chromosomes 1, 2, 4, 6, 7, 8, 14, 15, 16, 18, 19, 21, and 22 are reported. Two sSMC are neocentric and derived from 15q24.1-qter and 2q35-q36, respectively. Two further cases each present with two sSMC of different chromosomal origin. sSMC were characterized by multicolor fluorescence in situ hybridization for their chromosomal origin and genetic content. Moreover, uniparental disomy of the sister chromosomes of the sSMC was excluded in all nine cases studied for that reason. The 16 cases provide information to establish a refined genotype-phenotype correlation of sSMC and are available for future studies.

A rare case of chronic myeloid leukemia with secondary chromosomal changes including partial trisomy 17q21 to 17qter and partial monosomy of 16p13.3

BackgroundThe so-called Philadelphia (Ph) chromosome is present in almost all cases with chronic myeloid leukemia (CML). Around 5-10% of these patients show complex translocations involving other chromosomes in addition to and/or besides chromosomes 9 and 22. As nowadays most CML cases are treated with Imatinib, variant rearrangements have in general no specific prognostic significance, though events of therapy resistance remain to be studied.ResultsHere we report a Ph chromosome positive patient with hematological typical chronic phase CML. Untypically, an unbalanced complex rearrangement involving chromosomes 16 and 17 leading to a deletion of 16pter and partial trisomy of 17q21 to 17qter, was identified besides a trisomy 8 and an additional Ph chromosome in a part of malignant cells.ConclusionHere a novel and cytogenetically unique case of a Ph chromosome positive CML clinically in chronic phase is reported, having complex secondary chromosomal aberrations. Thus, CML patients with complex chromosomal changes are nonetheless treatable by Imatinib.

Secondary abnormalities involving 1q or 13q and poor outcome in high stage Burkitt leukemia/lymphoma cases with 8q24 rearrangement at diagnosis

Classical Burkitt lymphoma/leukemia (BL/L) presenting L3 morphology is found in 1% of childhood ALL. Recently, it has been described that secondary abnormalities could influence the prognosis of these patients. However, little information is available on these cytogenetic abnormalities and their prognostic importance in BL/L. Here, we report four new childhood BL/L cases associated with duplication within 1q or 13q, which exhibited a very unfavorable therapeutic response. We performed both classical and molecular cytogenetic analysis by multicolor chromosome banding of the secondary abnormalities involving the long arms of chromosome 1 or 13. These patients were previously treated with BFM-90 protocol. All of them died during or after the initial treatment. Here, for the first time, the exact breakpoints of the derivative chromosomes involved were determined at the cytogenetic level as 1q21 and 13q33 each.